Small non-coding microRNAs (miRNAs) regulate gene expression mainly at the posttranscriptional level. miRNAs play fundamentally important roles in various types of living organisms. Defects in miRNA-dependent gene regulation have been implicated in human diseases such as cancer and mental disorders. However, knowledge about functions of miRNAs in the mammalian skeletal system is limited. The goal of this research is to establish the roles of miRNAs during skeletal development in mice. miRNAs are generated through multiple processing steps; primary transcripts of miRNAs are first processed by the microprocessor containing Drosha and DGCR8 as critical components, and then by Dicer. Mature miRNAs are then loaded onto the RNA-induced silencing complex (RISC), that contains Argonaute (Ago) as the core component, and bind to their target transcripts through partial base-pairing. Our findings in Dicer- deficient mouse growth plates lead to our central hypothesis that chondrocytic miRNAs regulate cell proliferation and differentiation. However, because Dicer is known to process RNA species other than miRNAs, it is not clear to what extent the loss of miRNAs contribute to the phenotype of Dicer-deficient chondrocytes. In Aim I, we will confirm the role of miRNAs in regulation of chondrocyte proliferation and differentiation by generating and analyzing conditional knockout mice missing DGCR8 and Drosha to eliminate miRNAs independently of Dicer. In Aim II, to understand the mechanisms by which miRNAs regulate chondrocyte proliferation and differentiation, first we will experimentally identify miRNA-regulated transcripts in chondrocytes by profiling Ago2- associated RNAs. Second, proliferation defect of miRNA-deficient chondrocytes will be characterized by flow cytometry. In Aim III, we will investigate the physiological role of chondrocyte-specific miRNAs, miR-140 and miR-140*, in skeletogenesis. The Mir140 gene, encoding these miRNAs, is evolutionarily conserved in vertebrates, suggesting its important role in the skeletal system. Mice missing the Mir140 gene will be generated and characterized. Further, target transcripts of Mir140 miRNAs will be experimentally identified by the labeled miRNA pull-down assay (LAMP). Digoxigenin-labeled pre- Mir140 miRNA will be mixed with chondrocytes to reconstitute RISCs in vitro. Mir140 miRNA-bound transcripts will be immunopurified using anti-digoxigenin antibody. Defining roles of miRNAs and identifying their targets in chondrocytes may lead to identification of novel therapeutic targets for skeletal disease intervention. PUBLIC HEALTH RELEVANCE: The newly discovered gene regulatory mechanism through microRNAs has been shown to play important physiological and pathophysiological roles in a wide variety of organisms including humans. However knowledge about their roles in the skeletal system is very rudimentary. This proposal is aimed to establish the role of microRNAs in the skeletal system and to provide a basis for potential therapeutic interventions for skeletal diseases.